Targeted Identification of Rice Grain-Associated Gene Allelic Variation through Mutation Induction, Targeted Sequencing, and Whole Genome Sequencing Combined with a Mixed-Samples Strategy

Abstract Background: The mining of excellent allelic variation and the creation of new crop germplasm are the basis for improving breeding efficiency.Results: In this study, a total of 3872 heavy ion-irradiated M2 generation rice seeds and individual leaves were collected, and 484 mixed samples were obtained at a seed-to-leaf ratio of 8:1. Targeted sequencing technology was used to detect the major grain length genes GS3 and grain length. The allelic variation in the broad major gene GW5 and the phenotype data were measured, and 12 mixed samples containing potential mutations and 15 SNPs were obtained; 96 individual plants in the 12 mixed samples were then subjected to Sanger sequencing, combined with the phenotypic data obtained 13 key mutants and their corresponding SNPs; the protein structure and function analysis of the key mutants was carried out, and 6 allelic variants that led to the change in grain shape were mined, as well as the corresponding mutants, including long-grain mutants GS3-2 and GS3-7, short-grain mutants GS3-3 and GS3-5, wide-grain mutant GW5-1 and narrow-grain mutant GW5-4; sequencing identified new grain length gene allelic variants GS3-G1, GS3-G2 and GS3-G3, which provided new functional gene loci and a material basis for molecular breeding and genotype mutation and phenotype analysis, particularly in the context of mutational associations.Conclusion: Based on the above studies, we propose a method for targeted identification of allelic variation in rice grain type genes by combining targeted sequencing of mixed samples and whole genome sequencing. This method can sequence the target fragments of mixed samples, and the detection cost is low; furthermore, the identification cycle based on present method is short due to screening process is on the mutation offspring and the recombination genetic population is not necessary. The principle of present method is not limited to the grain type phenotype and can also be used for the directional detection and identification of other target traits and allele genes.